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Use of the ptxD gene as a portable selectable marker for chloroplast

transformation in Chlamydomonas reinhardtii

Article  in  Molecular Biotechnology · June 2019

DOI: 10.1007/s12033-019-00177-3

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Molecular Biotechnology
https://doi.org/10.1007/s12033-019-00177-3

ORIGINAL PAPER

Use of the ptxD gene as a portable selectable marker for chloroplast

transformation in Chlamydomonas reinhardtii
José M. Sandoval‑Vargas1 · Luis A. Jiménez‑Clemente1 · Karla S. Macedo‑Osorio1 · María C. Oliver‑Salvador1 ·
Luis C. Fernández‑Linares1 · Noé V. Durán‑Figueroa1 · Jesús A. Badillo‑Corona1

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that
can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas
reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering
to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use
of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across
life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element
that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to
oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two
positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes,
that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery
of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD
gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using
Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for
chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed
is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C.
reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering
in the chloroplast of C. reinhardtii.

Keywords  Phosphite assimilation · Chlamydomonas reinhardtii · ptxD · Selectable marker · Chloroplast transformation

Introduction C. reinhardtii has the potential to serve as the chassis for

Algae are photosynthetic eukaryotic organisms that natu-
rally produce a wide range of metabolites such as pigments,
lipids and polysaccharides. One in particular, the unicellular
green alga Chlamydomonas reinhardtii, has served both as
a model organism to study photosynthesis, phototropism,
flagellar function and cell cycle control and as a platform
to explore production of recombinant proteins, terpenoids
and hydrogen and lipids for biofuels [1–3]. Furthermore,
* Jesús A. Badillo‑Corona
jbadilloc@ipn.mx
1
Instituto Politécnico Nacional, Unidad Profesional
Interdisciplinaria de Biotecnología, Av. Acueducto SN, Col.
Barrio la Laguna Ticomán, 07340 Mexico City, Mexico
sustainable synthetic biology [4] to broaden the availability
and diversity of natural products, including enzymes, fatty
acids, pigments, vitamins, antibodies, therapeutic-drugs,
food additives, polysaccharides, polymers and biomass.
Engineering of the nuclear and chloroplast genomes is pos-
sible in C. reinhardtii, but the latter is the technology, known
as transplastomic technology, that has emerged as promising
alternative to overcome many of the drawbacks associated
with nuclear transformation.
For the last two decades, transplastomic technology in C.
reinhardtii has been mostly used to demonstrate that high-
value and complex compounds such as antibodies, therapeu-
tic proteins and vaccines can be produced at economically
feasible levels [5, 6]. More recently, efforts have turned to
explore the feasibility of using this alga as a platform for

13
Vol.:(0123456789)

the production of commodities that could be used to address
the demand of biofuels [3, 7]. The chloroplast of C. rein-
hardtii contains ~ 80 copies of a 205-kb genome compris-
ing each over 100 genes coding for proteins and RNAs that
are involved in photosynthesis and plastid transcription and
translation [8, 9]. To achieve chloroplast transformation,
selectable marker genes are essential tools for efficient iden-
tification of transformed strains. Chloroplast transformation
was first achieved in C. reinhardtii by restoring photosyn-
thesis following introduction of the wild-type aptB gene into
mutant strains carrying deletions or point mutations in this
gene [10]. Since then, use of the corresponding wild-type
gene to restore photosynthesis in recipient lines carrying
large deletions, frameshift mutations or non-sense deletions
in the psbA [11], psaB [12], tscA [13], atpE [14] and rbcL
[15] genes has been successfully demonstrated. However,
these approaches have relied on the use of existing mutant
from forward genetic screenings. More recently, the group
of Saul Purton have developed a strain in which the psbH
gene has been disrupted with an expression cassette for the
aadA gene [16, 17]. This photosynthetic deficient strain can-
not grow in the absence of acetate. Transformation in this
strain can be then accomplished with the use of a vector car-
rying the gene of interest and a functional psbH gene [18].
After recombination, the aadA gene is eliminated yielding
photosynthetically competent and aadA-free cells. This
marker-free approach has been used to express the PfCel-
TOS antigen from P. falciparum to develop an ELISA test
for diagnosis of malaria [19] and the fusion protein con-
sisting of the PfCelTOS antigen and human interleukin-2
(PfCelTOS:IL-2), a vaccine candidate fused to human inter-
leukin-2 (IL-2) as vaccine adjuvant [20].
In contrast to the more diverse options available for chlo-
roplast transformation in plants [21–23], chloroplast trans-
formation in C. reinhardtii currently counts with just two
portable selectable markers, that is, transgenes that coupled
with endogenous cis transcription and translation elements
can be used for direct recovery of transformed lines based
on a phenotypic trait. These dominant antibiotic resist-
ance markers are: the aadA gene [13], encoding an ami-
noglycoside 3″-adenylyltransferase conferring resistance
to spectinomycin, and the aphA-6 gene [24], encoding an
aminoglycoside 3′-phosphotransferase conferring resistance
to kanamycin. These markers have been used extensively
with a low, but relevant, false positives rate; up to 10% for
the aphA-6 gene [24]. Given the concerns of using genes
that confer resistance to antibiotics and the strict regulatory
requirements for the commercialization of genetically modi-
fied organisms and their products, development of efficient,
portable selectable markers that can spare the use of antibi-
otics is highly needed.
Recently, we have shown that the ptxD gene can be
used to engineer the chloroplast of C. reinhardtii to enable
13
Molecular Biotechnology
phosphite assimilation [25]. The ptxD gene, from the ptxAB-
CDE operon from Pseudomonas stutzeri WM 88, encodes a
NAD-dependent phosphite dehydrogenase, which catalyzes
the oxidation of Phi to Pi with the concomitant reduction
of ­NAD+ to NADH [26]. Following stable integration and
expression of the ptxD gene, strains of C. reinhardtii were
able to assimilate phosphite and use it as the sole phospho-
rus source. However, in our previous work we had to select
transformed lines based on their ability to tolerate the pres-
ence of the antibiotic spectinomycin, as the transformation
of chloroplasts was carried out in combination with vec-
tor p228 (Chlamydomonas Resource Center, University
of Minnesota) which carries a dominant point mutation
(spr-u-1-6-2) in the 16S rRNA gene [27]. This had to be
done because we did not know if transformed lines could be
directly selected based on their ability to metabolize phos-
phite. Here we investigated if the ptxD gene could be used
directly, without the use of genes that confer resistance to
antibiotics, as a selectable marker to carry out chloroplast
transformation in C. reinhardtii. We are showing that trans-
formed cells develop into visible colonies when growing in
media containing phosphite as the sole phosphorus source
with no false positive colonies. Use of the ptxD gene as
selectable marker for nuclear transformation of C. rein-
hardtii has already been demonstrated, but this is the first
time that this gene has been used as selectable marker for
chloroplast transformation in this photosynthetic organism.
Furthermore, we are demonstrating that phosphite-assimi-
lating strains can endure relaxed conditions of media and
photobioreactor preparation with no apparent contamination
during a 15-day cultivation time-course.
Materials and Methods
Algal Strain and Culture Conditions
The wild-type C. reinhardtii CC-125 (mt +) strain (Cr-wt)
was obtained from the Chlamydomonas Resource Center
(Chlamydomonas Resource Center, University of Min-
nesota, USA) and grown in standard TAP medium [28].
Additionally, four media, termed TA, TAPhi, TP and TPhi,
were used at the same pH and molar concentrations of com-
ponents as in the TAP formulation. TA medium contains
no phosphorus source; TAPhi medium contains monobasic
potassium phosphite (­ KH2PO3; Wanjie International, China),
whereas TP and TPhi media contain no acetate as carbon
source and contain phosphate and phosphite as phosphorus
source, respectively. For solid cultures, 1.5% (w/v) of agar
was used. Solid cultures were maintained under illumination
at 50 µmol photons ­m−2 s−1 of white LED light, in a 16:8 h
(light/dark) photoperiod at 25 °C while sterile liquid cultures
were grown under the same conditions but with constant
Molecular Biotechnology
agitation at 150 rpm in an orbital shaker. For P-starved
cultures, cells at late mid-log phase in TAP medium were
harvested and washed twice with TA medium. A total of
1 × 108 cells were transferred to liquid TA medium for 7 or
14 days with agitation in an orbital shaker at 150 rpm and
under the same culture conditions as described above. After
each starvation period, cells were harvested, washed twice
with TA medium, and re-suspended in TA to reach a final
concentration of 1 × 105 cells.
Bottles (1 L) containing 800 mL of non-sterile TP, for
the wild-type strain, and 800 mL of non-sterile TPhi, for
transplastomic strains, were inoculated with 1­ x105 TAP-
grown cells, placed in a 12:12 h (light–dark) photoperiod
at 24° C under 180 μmol photons m ­ −2 s−1 of white light and
air-bubbled at 0.4–0.5 vvm. The algal growth was estimated
every 24 h by measuring the optical density at 750 nm and
by direct cell counting using a hemocytometer.
Chloroplast Transformation
Chloroplast transformation was carried out by particle bom-
bardment using p320-PpsbD-ptxD according to the meth-
odology described in [29] with slight modifications. Prior
to bombardment, 7-days or 14-days P-starved wild-type C.
reinhardtii cells were plated over solid 5 mM TAPhi media
and placed under sterile conditions in a dark room for 2 h
to air-dry the excess of liquid medium. Plated cells were
subjected to particle bombardment with the Biolistic PDS-
1000/He Particle Delivery System (Bio Rad) at 9 cm of dis-
tance using 1100 psi rupture disks (Bio Rad) and 30 in Hg
vacuum pressure within the chamber. After bombardment,
plates were maintained in the dark for 3 days before being
transferred to an incubation chamber at 25 °C with a 16:8 h
light/dark photoperiod for > 7 weeks. TAPhi-resistant colo-
nies were selected among the green lawn and transferred
to freshly prepared TAPhi plates and sub-cultivated for
1–2 weeks.
Molecular Characterization
DNA extraction from wild-type and transplastomic lines was
carried out using the resin Chelex-100 (Bio-Rad) follow-
ing a previously reported method [29, 30]. PCR was carried
out using PCR 2 × Master mix (Thermo- Scientific, USA)
following the manufacturer’s instructions using JAB489
(5′-ATG​GAC​TAT​AAG​GAT​CAC​GA-3′) and JAB490 (5′-
TTA​GCA​GGC​GGC​GGG​CTC​CG-3´) primers to amplify
the ~ 1100 bp product corresponding to the ptxD gene, and
primers LCT 59 (5′-CGTG ​ GAG​ TAT
​ TTA​ ATA​ CAG
​ CTA-3′)
and LCT 68 (5′-CCC​ATA​AAT​AGT​TTC​AAT​TGGA-3′) to
amplify the 2174-bp product corresponding to the PpsbD-
ptxD-TrbcL cassette. The PCR products were analyzed by
gel electrophoresis (1% agarose) using Gel Red DNA Gel
Stain (Biotium Inc, USA).
Immunodetection of PTXD
Total soluble protein (TSP) extraction was carried out
according to [11]. TSP was quantified by Bradford assay
with Quick Start Bradford 1 × Dye Reagent (Bio-Rad) fol-
lowing the manufacturer’s instructions and using bovine
serum albumin as a standard for a calibration curve of known
amounts. A total of 30 μg of TSP were separated by electro-
phoresis in a 12% polyacrylamide gel with denaturing condi-
tions and then transferred to Immun-Blot PVDF Membranes
(Bio-Rad) as described by [31]. Membranes were probed
with a horseradish peroxidase conjugated mouse monoclonal
antibody to the DDDDK epitope present in the 3X-FLAG
tag at the N-terminal of PTXD (Abcam, United Kingdom).
The membrane was developed with Western Lightning Plus-
ECL reagent (Perkin Elmer Inc., Netherlands) and recorded
using a Chemi DOC MP Imaging system (Bio Rad).
Southern Blotting
For Southern blotting, total DNA from wild-type and
transplastomic C. reinhardtii was isolated using the Wiz-
ard Genomic DNA Purification Kit (Promega, USA) fol-
lowing the manufacturer’s instructions for plant extraction.
For DNA preparation, total DNA lines were digested with
endonucleases EcoRI and XhoI (Thermo Fisher Scientific,
USA). Fragments were separated by electrophoresis in a
0.8% agarose gel, and DNA was transferred to a Zeta-probe
nylon membrane (Bio Rad) by alkaline blotting following
the manufacturer’s instructions or according to [32]. Mem-
branes were hybridized with a DNA probe for the ptxD gene
(~ 1100 bp), which was amplified by PCR using the specific
primers (JAB489 and JAB490) and labeled with [α-32P]
dATP by random priming according to [33]. The membrane
was hybridized and exposed to a phosphor imaging screen
(Bio Rad) for 1–2 h. Screens were scanned using a Personal
Molecular Imager PMI (Bio Rad). The analysis of the image
obtained was done using the Quantity One 1-D Analysis
Software (Bio Rad).
Results and Discussion
To investigate if the ptxD gene can be directly used as a
selectable marker for chloroplast transformation in C. rein-
hardtii, we used vector p320-PpsbD-ptxD. In this vector,
the ptxD from P. stutzeri WM88 is under the control of the
psbD promoter and the rbcL terminator. The ptxD gene
was optimized for C. reinhardtii chloroplast codon usage
(Genbank MG823244) and contains the sequence coding
13

for a modified 3X-FLAG at the 5´-end. Flanking sequences
target the insertion of the expression cassette to the region
in the inverted repeat of the chloroplast genome between
23S-5S rRNA/exon5-intron 4 of psbA (Fig. 1a). As a first
approach, we followed the transformation protocol we
routinely use for chloroplast transformation using tung-
sten particle bombardment. Cells grown in TAP medium
were placed as a lawn on TAPhi medium, instead of the
antibiotic-supplemented TAP medium, normally used for
selection when the aadA or the aphA-6 genes are used
[29]. Following bombardment, we could not recover Phi-
assimilating colonies; in fact, we continued to observe a
grainy slightly pale green lawn of algae even after more
than 60 days in TAPhi media (Fig. 1b; event 1). This is
not entirely surprising, as it has been demonstrated that
Fig. 1  Generation of transplastomic, phosphite-assimilating lines of
C. reinhardtii using the ptxD gene as a selectable maker. a C. rein-
hardtii wild-type genome targeted for insertion of the ptxD gene
and chloroplast transformation vector p320-PpsbD-ptxD. Expres-
sion of ptxD gene is regulated by psbD promoter and rbcL termi-
nator, this expression cassette is flanked by homologous recom-
bination sequence of 23S-5S rRNA and exon 5-intron 4 of psbA. b
Particle bombardment of P-replete (Event 1) and 7-day P-depleted
13
Molecular Biotechnology
Phi does not support growth but is not toxic to algae, thus
allowing cells to remain viable using internal Pi reserves
[34]. We then reasoned that a stringent selective pressure
could favor the proliferation of ptxD transformed lines.
To do this, we cultivated wild-type cells in P-depleted
TAP media (termed here TA media) for 7-days with the
aim of starving the cells for P and force them to consume
endogenous Pi reserves. Upon introduction of the ptxD
gene, we thought, transformed cells would rapidly pro-
liferate whilst the starved cells would continue to die.
P-starved cells were used for transformation with vector
p320-PpsbD-ptxD. After about 50 days, we observed the
proliferation of a single colony growing in TAPhi media,
presumably using Phi as P source. When the colony was
about 5 mm (60 days post-bombardment), we transferred it
cells (Event 2). c Molecular characterization of the only phosphite
assimilating line Crc-PhiX obtained from bombardment of a 7-day
P-depleted culture. From top to bottom: PCR amplification of the
ptxD gene, RFLP analysis using a specific [α-32P] dATP labeled
probe specific for the full length ptxD coding sequence, immunode-
tection of the PTXD protein using an anti-3XFLAG monoclonal anti-
body and SDS-PAGE stained with Coomassie brilliant blue to verify
equal amount of protein loading
Molecular Biotechnology
to fresh TAPhi media (using a wild-type strain as a nega-
tive control) and allowed it to proliferate for a few days
(Fig. 1b; event 2). We termed this line Crc-PhiX (Chla-
mydomonas reinhardtii chloroplast phosphite assimilating
strain X) and carried out a full molecular characterization.
We found that the expression cassette had integrated in
the chloroplast genome in the expected locus and detected
the presence of the ptxD transcript and the 39-kDa PTXD
protein (Fig. 1c). We used one of our previously reported
Phi-assimilating strains Crc-Phi1 [25] and showed that, at
the molecular level, Crc-PhiX was indistinguishable from
Fig. 2  Optimization of the efficiency on the use of the ptxD gene
as a selectable marker for chloroplast transformation and growth of
phosphite-assimilating strains under non-sterile conditions. a Parti-
cle bombardment of 14-day P-depleted cells (Events 3 and 4). b PCR
amplification of the ptxD gene in lines obtained in Events 3 and 4.
PCR amplification of the 2174-bp PpsbD-ptxD-TrbcL cassette in
selected transplastomic lines and the 202-bp fragment in the wild-
Crc-Phi1, confirming that the ptxD can be directly used as
a portable selectable marker for chloroplast transformation
in C. reinhardtii.
Next, we tried to increase the transformation efficiency of
one Phi-assimilating colony per bombardment. We investi-
gated if an increase on the P-starvation period could have a
positive effect on the number of transformed lines recovered.
To do this, we used 14-day P-starved cells and followed the
same transformation protocol as described before. We found
that this time a greater number of colonies proliferated on
TAPhi media and were fully visible by day 30 (Fig. 2a).
type line to demonstrate homoplasmy. c Summary of transformation
events, P-starvation regime and transformation efficiencies. d Wild-
type strain in TP medium and phosphite-assimilating transplastomic
strains of C. reinhardtii, Crc-PhiX and Crc-Phi1, growing in non-ster-
ile photo- and mix-autotrophic conditions in TPhi and TAPhi media,
respectively. e Growth kinetics of transplastomic Crc-PhiX and Crc-
Phi1 lines growing in photo- and mix-autotrophic conditions
13

We carried out this experiment in duplicate and obtained an
average of 22 Phi-assimilating colonies (Fig. 2a), a consider-
able improvement from the one colony obtained with 7-day
P-starved cells (Fig. 2c). All colonies were used to deter-
mine the presence of the ptxD gene using specific primers
to amplify the 1100-bp ptxD gene. In all transformed lines,
but not in the wild-type strain, we successfully amplified a
fragment of the expected size (Fig. 2b, top two panels), indi-
cating that the ptxD gene was present in the lines obtained
and thus indirectly demonstrating that the Phi-assimilating
phenotype was the result of the biologically active PTXD
protein, catalyzing the oxidation of Phi to metabolizable
Pi. Notably, no false positives were detected, a drawback
frequently observed when the aadA [13] and aphA-6 genes
are used as selectable markers [24]. In all 11 lines obtained
in event 3, we determined homoplasmy by amplifying the
PpsbD-ptxD-TrbcL cassette using primers laying outside of
the BamHI and SpeI site in the wild-type and transformed
genome (Fig. 1a). We amplified the expected fragments of
202 bp and 2174 bp for the wild-type and transplastomic
lines, respectively. We did not get amplification of the 202-
bp fragment in the transformed lines, indicating that lines
were homoplasmic and that the wild-type genome was no
longer present. Figure 2c shows a summary of the results of
the transformation events we have described above. The effi-
ciency of transformation we have obtained (1-50 colonies) is
in the range of what is normally observed for other selecta-
ble markers, 5-100 transformed colonies for aphA-6 [24]
and 8-50 for 16S rRNA carrying a dominant point mutation
[35]. Other groups have used the ptxD gene as a portable
selectable marker, albeit for transformation of the nuclear
genome, in yeasts [36] and in plants including tobacco and
Arabidopsis [37], maize [38], cotton [39] and sorghum [40].
To the best of our knowledge, this is the first time that the
ptxD gene has been used as a portable selectable marker for
chloroplast transformation in C. reinhardtii, allowing direct
selection of transplastomic cells which are able to use phos-
phite as the phosphorus source.
Engineering phosphite metabolism in algae, and per-
haps other organisms, represents an invaluable tool for the
management of biological contaminants in closed and open
cultures [41] and to develop synthetic organisms that can be
self-contained [42]. Using nuclear transformed algae Loera-
Quezada et al. [41] have demonstrated that when cultivated
in bioreactors prepared with non-sterile water and chemicals,
Phi-assimilating strains can colonize a non-sterile environ-
ment where phosphite is the only source of phosphorus.
Furthermore, these strains can outcompete other species of
algae uncapable of assimilating Phi. Using one of our pre-
viously obtained transplastomic strains, Crc-Phi1 [25] and
Crc-PhiX (this work), we investigated if these lines, express-
ing the ptxD gene from the chloroplast genome, showed a
similar behavior to those reported by [41] when growing
13
Molecular Biotechnology
under photoautotrophic conditions in loosely prepared 1 L
cultures. Bottles containing non-sterile TPhi media were
inoculated with 1­ x105 TAP-grown cells, placed in a 12-h
light/12-h dark photoperiod under 180 μmol photons/m2·s
and air-bubbled at 0.4–0.5 vvm without the use of air fil-
ters (Fig. 2d). We measured the cell density daily and made
observations under the microscope to follow the evolution
of the culture. Both Crc-Phi1 and Crc-PhiX growing in
TPhi media remained without noticeable contamination up
to 15 days after the start of the culture and reaching a cell
density of ~ 1.6 × 106 cell/mL (Fig. 2e, TPhi-CrcPhiX and
TPhi-Crc-Phi1) similar to a wild-type strain growing in TAP
media under sterile conditions (not shown). By contrast, the
culture inoculated with the wild-type strain, growing in TP
media, was overgrown by invading species by day 7 (Fig. 2d,
TP-Wild-type; Fig. 2e, TP-Cr-Wt). Similarly, the Crc-PhiX
strain, growing in TAPhi media was also overgrown by uni-
dentified invading species of bacteria and fungi revealed
by observations we made under the microscope (Fig. 2d,
TAPhi-Crc-PhiX). We did not follow-up to identify the
invading species as it falls out of the scope of this study.
Our results show that transplastomic strains of C. reinhardtii
expressing the ptxD gene can be grown under non-sterile
condition, keeping species that are uncapable of assimilating
phosphite at bay. Furthermore, we have kept lines Crc-Phi1
and Crc-PhiX for 2 years and 6 months, respectively, grow-
ing in phosphite as the sole phosphorus source and have not
detected signs of the ptxD gene being unstable.
Our result is highly relevant as it opens the possibility
to use chloroplast transformed strains of a generally rec-
ognized as safe (GRAS) organism for the production of
biotechnologically, perhaps non-health-related, compounds
in laxly prepared semi-closed bioreactors or open raceway
reactors. These type of cultures would keep the overall pro-
duction cost low, as the requirement for the sterilization of
upstream unitary operations could be eliminated. Phosphite
assimilating cultures producing industrially relevant metabo-
lites could be kept for longer thus increasing the productiv-
ity while lowering the cost of the final product. Recently
cyanobacteria have been engineered to rely exclusively on
the assimilation of phosphite for growth [42], as opposed to
our strains, which can also use phosphate, suggesting that a
similar strategy could be applied to an eukaryotic alga such
as C. reinhardtii in the future.
Conclusions
Here we have shown that the ptxD gene can be used as a
selectable marker for chloroplast transformation in Chla-
mydomonas reinhardtii allowing easy visual selection of
phosphite-assimilating colonies without the need of antibi-
otics. Furthermore, we have shown that the transformation
Molecular Biotechnology
efficiency increases when 14-day P-starved cells are used
for transformation and that, importantly, no false positives
arise. We have also demonstrated that chloroplast trans-
formed, phosphite-assimilating strains of C. reinhardtii can
grow under non-sterile conditions in semi-closed 1-L liquid
air-bubbled glass bottles without noticeable invasion from
bacteria and fungi. The ptxD gene as a portable selectable
marker we have developed arrives as a relevant tool more
than 18 years after the last one (aphA-6) was developed.
Our selectable marker expands the tool box for chloroplast
genetic and metabolic engineering in C. reinhardtii.
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